The present invention relates to the improvement of hydrogen production by Chlamydomonas reinhardtii. 
Hydrogen is an essential starting material for the chemical industry, and also constitutes a fuel called upon to play a major role in the coming decades. Hydrogen-fed fuel cells make it possible, through the reaction of hydrogen with oxygen from the air, to produce electricity in a nonpolluting manner, while producing only steam as waste. The technological advances in the field of fuel cells make their use on a large scale increasingly foreseeable.
However, the majority of the hydrogen currently used is produced from fossil energy sources, such as petroleum or carbon, by techniques that themselves generate pollution, such as catalytic conversion of hydrocarbons from natural gas or cracking petroleum or carbon.
It therefore appears desirable to provide cost-effective processes for producing hydrogen from a renewable and clean primary energy source (that does not release greenhouse gases).
Certain unicellular green algae belonging to the genera Scenedesmus, Chlorococcum, Chlorella (order Chlorococcales), Lobochlamys and Chlamydomonas (order Volvocales), such as the species Chlamiydomonas reinhardtii, are capable of producing hydrogen from solar energy, using water as electron and proton donor.
In these algae, the hydrogen is produced by an iron hydrogenase with a strong specific activity. This enzyme is connected to the PSI photosynthetic electron transfer chain via a common electron transporter, ferredoxin. The electrons required for the production of hydrogen can be supplied to PSI either through the activity of PSII (pathway A), or through the use of carbon stores via the nonphotochemical reduction of plastoquinones (pathway B). These two pathways are shown schematically in FIG. 1.
Legend of FIG. 1: In solid lines, pathway A, PSII-dependent; in dashed lines, pathway B, based on plastoquinone reduction, PSII-independent.
PSI: photosystem I; PSII: photosystem II; RuBP: ribulose 1,5-bisphosphate; LHC: light harvesting complex; FNR: ferredoxin NADP reductase; Fd: ferredoxin; Pc: plastocyanin; cytb6: cytochrome b6; cytf: cytochrome f; NDH: NADH-dehydrogenase; PQ(H)2: plastoquinol; Qa: quinone a; P680 and P700: reaction centers for PSI and PSII, respectively.
Under natural conditions, H2 production is only a transient phenomenon. In fact, the hydrogenase is very sensitive to O2. Now, the photolysis of water occurring within photosystem II, which supplies electrons for the production of H2 via pathway A, also produces O2, which induces a rapid inhibition of the hydrogenase.
Various solutions have been proposed in order to remedy this problem. The first solution is based on production in the dark, the others on production under light using in part pathway B, which, unlike pathway A, does not lead to oxygen production.
For example, U.S. Pat. No. 4,532,210 describes a process alternating light and dark phases. During the light periods, the algae produce O2 and accumulate hydrocarbon-based stores produced by photosynthesis. These stores are then used under anaerobic conditions during the dark phases in order to produce hydrogen. This method requires a nitrogen purge in order to achieve the anaerobiosis. It is also limited by the efficiency of H2 production in the dark, which is an order of magnitude lower than production under light.
U.S. application 2001/0053543 describes a process based on the reversible inhibition of photosystem II by means of a sulfur deficiency. This process comprises a step consisting in culturing green algae, under light, and in a medium with a normal sulfur content, so as to allow the accumulation of hydrocarbon-based stores, and a step consisting in culturing in sealed containers, and under light, in a medium lacking sulfur. The inhibition of photosystem II leads to an arrest of oxygen production by photosynthesis. When the algae (in which respiration is not inhibited by the sulfur deficiency) have used all the oxygen present in the medium, they become anaerobic and use the hydrocarbon-based stores produced due to photosynthesis, to produce H2. Alternation of phases of culturing in the presence and absence of sulfur makes it possible to temporally separate the light phases of O2 production and H2 production.
U.S. application 2003/0162273 proposes an alternative method for inducing a sulfur deficiency that inhibits photosystem II; it involves the use of a genetically modified alga underexpressing a chloroplast sulfate permease.
The methods described above make it possible to prevent inhibition of the iron hydrogenase by separating the production of O2 and that of H2. However, the production of hydrogen by the three methods described above has limitations. The first method is based on the fermentative activity of the alga in the dark, a relatively ineffective phenomenon that results in only marginal production of hydrogen. The second and third methods are based on the parallel functioning of pathways A and B. Pathway A, which is accompanied by oxygen release, must be maintained at a level below respiratory O2 consumption in order to maintain anoxia. Pathway A is therefore limited by the respiratory capacity of the algae. The contribution of pathway B is significant but limited.
The aim of the present invention is to improve the yield of this second pathway (B). With this aim, the inventors had the idea of using, in the chloroplast, a type II NADH-dehydrogenase for stimulating the plastoquinone reduction reaction.
Type I and II NADH dehydrogenases are enzymes capable of reducing the quinones of electron transport chains. They are associated with mitochondrial and bacterial respiratory chains (KERSCHER, Biochim. Biophys. Acta 1459: 274-283, 2000).
Type I NADH dehydrogenases (NDH-I) are multimeric transmembrane complexes comprising from 14 to approximately 50 subunits. This type of complex oxidizes only NADH and has an associated proton pumping activity.
Type II NAD(P)H dehydrogenases (NDH-II) are monomeric enzymes of oxidoreductase type, which have a molecular weight of between 30 and 60 kDa and are capable of reducing the quinones of bacterial respiratory chains or mitochondrial chains of plants and yeasts, by oxidizing NADH or NADPH. Their association with the photosynthetic chains of plants and algae has also been proposed, but has not been demonstrated to date. This type of enzyme has not been demonstrated in the animal kingdom.
In the chloroplasts of higher plants, the existence of a functional NDH-I complex has been demonstrated (BURROWS et al., EMBO J. 17: 868-876, 1998; SAZANOV et al., Proc. Natl. Acad. Sci. USA 95: 1319-1324, 1998; HORVATH et al., Plant Physiol. 123: 1337-1349, 2000) and the existence of an NDH-II-type activity has also been proposed (CORNEILLE et al., Biochim. Biophys. Acta 1363: 59-69, 1998). In Chlamydomonas reinhardtii, the genes encoding the chloroplast NDH-I complex are absent. However, existence of an NDH-II-type activity has been suggested (COURNAC et al., Int. J. Hydrog. Energy 27: 1229-1237, 2002; PELTIER and COURNAC, Annu. Rev. Plant Biol. 53: 523-550, 2002).